Bionectria ochroleuca is potentially useful for bio-control. The strain B. ochroleuca YLZ42 isolated from the soil in Yulin of the Guangxi Zhuang Autonomous Region of China was previously shown to be able to kill various agricultural pests, including the diamondback moth, Plutella xylostella. This study aimed to isolate the insecticidal metabolite from the fermentation broth of B. ochroleuca YLZ42 and study its biological activity and stability. The isolation of the insecticidal metabolite was based on a bioassay-guided method. As a consequence, the optimized brine shrimp lethality test (BSLT) was chosen to evaluate the metabolite’s insecticidal ability. Silica gel column chromatography, extraction, preparative thin layer chromatography (TLC), and reverse-phase high-performance liquid chromatography (HPLC) were conducted to isolate the insecticidal metabolite. After isolation, the biological activity and stability of this insecticidal metabolite were then studied. Furthermore, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and proton nuclear magnetic resonance (1H-NMR) were used to further analyze the metabolite. A pure metabolite with insecticidal activity was obtained. The lethal concentration (LC50) of this metabolite against brine shrimp was found to be 86.89 mg/L after 24 h post-treatment. In addition, the stability of this insecticidal metabolite indicated that its insecticidal activity was stable while the temperature was below and the pH was between 6 and 8. Further, the metabolite was not sensitive to light in the UV to visible range. The molecular weight of this compound was determined with HR-ESI-MS to be 788. The 1H-NMR spectrum of this insecticidal metabolite was different from that of any other reported compound with the same molecular weight. The insecticidal metabolite has the potential to be developed as a new microbe-derived pesticide.